阅读说明：本技术 芝麻蛋白SiBRB在调控植物根系发育中的应用 (Application of sesame protein SiBRB in regulation and control of plant root system development ) 是由 游均 山姆 苏如齐 黎冬华 张秀荣 周瑢 刘爱丽 于 2020-07-08 设计创作，主要内容包括：本发明阐述了芝麻蛋白SiBRB在调控植物根系发育中的应用。通过将芝麻蛋白SiBRB的编码基因在拟南芥中的过表达,发现此基因可显著减少拟南芥植株的总侧根长度和根鲜重,最终可以减少根系总量。依此表明,该基因是限制植株根系生长的关键基因,在调控植物根系发育、调节植物根冠比中有很好的应用前景。(The invention discloses application of sesame protein SiBRB in regulation and control of plant root system development. Through the overexpression of the encoding gene of the sesamin SiBRB in arabidopsis thaliana, the gene is found to be capable of remarkably reducing the total lateral root length and the fresh root weight of arabidopsis thaliana plants, and finally the total root system amount can be reduced. Therefore, the gene is a key gene for limiting the growth of plant root systems, and has good application prospects in regulating and controlling the development of plant root systems and regulating the ratio of root crowns of plants.)

1. The application of the sesame protein SiBRB or the related biological material thereof in regulating and controlling the development of plant roots; the related biological material is a nucleic acid molecule capable of expressing the sesamin SiBRB or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

2. The use according to claim 1, wherein the sesame protein SiBRB or a gene encoding the same is expressed in the plant in an increased amount and/or activity, which reduces root development in the plant.

3. Use as claimed in claim 1 or claim 2 wherein the plant root development is manifested as at least one of: the total lateral root of the plant is long and the root is fresh and heavy.

4. The use according to claim 1 or 2, wherein the sesamin SiBRB is an amino acid sequence having an amino acid sequence shown in SEQ ID No.1 or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids in the sequence.

5. The use according to claim 1 or 2, wherein the encoding gene of the sesamin SiBRB is a DNA sequence as shown in SEQ ID No.2 or a DNA sequence with equivalent function generated by adding, substituting, inserting or deleting one or more nucleotides in the sequence.

6. A method for breeding a plant variety having reduced total lateral root length and/or reduced fresh root weight, comprising the step of increasing the expression level and/or activity of sesamin SiBRB as claimed in claim 1 in a recipient plant.

7. A method of breeding transgenic plants with reduced total lateral root length and/or reduced fresh root weight comprising the steps of: introducing into a recipient plant a nucleic acid molecule capable of expressing sesamin SiBRB as claimed in claim 1, resulting in a transgenic plant; the transgenic plant has reduced total lateral root length and/or reduced fresh root weight as compared to the recipient plant.

8. The method of claim 7, wherein said introducing into a recipient plant a nucleic acid molecule capable of expressing sesamin SiBRB is effected by introducing into said recipient plant a recombinant expression vector comprising a gene encoding said sesamin SiBRB.

9. The method of claim 7, wherein the recipient plant is Arabidopsis thaliana.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to application of sesame protein SiBRB in regulation and control of plant root system development.

Background

Sesame (Sesamum indicum L.) is one of important high-quality oil crops in the world, the annual planting area of Chinese sesame is 80 ten thousand hectares, the total yield is about 75 ten thousand tons, and the sesame occupies the top of the world. Meanwhile, sesame is a typical oil crop planted in summer, is warm and bright, and can complete the whole growth period within 3-4 months. Despite the many advantages of sesame, the problems of low and unstable sesame yield have been significant problems limiting the development of the sesame industry. The main reason for the limited sesame production is that sesame is mainly distributed in developing countries, usually in small farmers. Although sesame has a high yield potential, the actual yield is rather low and unstable due to the combined action of biotic and abiotic stress, which is also a significant bottleneck problem for the development of the sesame industry.

Sesame is a shallow root crop, the sesame root system is not only an organ for supporting the plant body to carry out vegetative growth, but also a main organ for absorbing water and mineral elements, and is also a metabolic circulation organ, and the sesame is sensitive to the response of external environmental conditions. The sesame root system is divided into a straight root system and a branch root system, wherein the straight root system is characterized in that the main root is very long and the side roots are few; the branch root system has less developed main root and more lateral roots. Generally, sesame varieties in arid areas are mostly straight root systems, and sesame varieties in sandy soil or humid areas are mostly branch root systems. However, the root system of sesame is an underdeveloped root system, and the total amount of the root system is less, so that the improvement of the yield of a single sesame plant is indirectly influenced.

Disclosure of Invention

The invention aims to provide application of sesame protein SiBRB in regulation and control of plant root system development. The sesame protein SiBRB and the coding gene thereof have very low homology with arabidopsis thaliana and other crops, and the gene sequence has no functional annotation, the invention proves that the gene and the expression protein thereof have the function of reducing the total amount of the root system of the plant for the first time, the gene can be transferred into a receptor plant through a gene engineering technology to achieve the purposes of reducing the root system and regulating the root-crown ratio, and the gene can also be mutated through a gene editing means to achieve the purpose of improving the total amount of the root system of the plant.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the application of the sesame protein SiBRB or the related biological material thereof in regulating and controlling the development of plant roots; the related biological material is a nucleic acid molecule capable of expressing the sesamin SiBRB or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

A method for producing a plant variety having reduced total lateral root length and/or reduced root fresh weight, comprising the step of increasing the expression level and/or activity of said sesamin SiBRB in a recipient plant.

A method of breeding transgenic plants with reduced total lateral root length and/or reduced fresh root weight comprising the steps of: introducing a nucleic acid molecule capable of expressing the sesamin SiBRB into a receptor plant to obtain a transgenic plant; the transgenic plant has reduced total lateral root length and/or reduced fresh root weight as compared to the recipient plant.

Compared with the prior art, the invention has the advantages that:

the invention can reduce the total root system amount of a receptor plant through overexpression by transferring the nucleic acid molecule capable of expressing the sesame protein SiBRB into the receptor plant. According to the method provided by the invention, the over-expression of the sesame protein SiBRB can be utilized to regulate and control the development of the plant root system and the ratio of the root to the crown of the plant.

Drawings

FIG. 1 is a diagram of the construction of plant recombinant expression vector pCAMBIA1301S-SiBRB containing SiBRB gene provided in the embodiment of the present invention;

FIG. 2 is a screening plate of transgenic SiBRB gene in Arabidopsis thaliana T1 generation plant provided by the embodiment of the present invention; in the figure, the left figure is a whole flat plate figure, and the right figure is a partial enlarged view;

FIG. 3 is a schematic diagram of the results of PCR identification of SiBRB transgenic Arabidopsis thaliana generation T1, wherein a lane M indicates Marker, lanes 1-4 are transgenic plants of generation T1, and a lane WT is wild Arabidopsis thaliana;

FIG. 4 is a plate diagram of a screen of transgenic SiBRB gene in Arabidopsis thaliana T2 generation plants, in which the left panel is the whole plate diagram and the right panel is a partial enlarged view;

FIG. 5 is a schematic diagram of the results of PCR identification of transgenic SiBRB Arabidopsis thaliana generation T2 provided in the example of the present invention, in which lane M represents Marker, lanes 1-6 are transgenic plants of generation T2, and lane WT is wild Arabidopsis thaliana;

FIG. 6 is a comparison of the results of the quantitative expression verification of the SiBRB transgenic Arabidopsis T2 generation plant groups (SiBRB-1, SiBRB-2 and SiBRB-3) and the wild type Arabidopsis plant group (WT) according to the present invention;

FIG. 7 is a scanned map of the root system of an Arabidopsis plant according to the present invention, wherein the left image is a wild plant and the right image is a SiBRB transgenic plant;

FIG. 8 is a graph comparing the results of the total lateral root length (cm) of the SiBRB transgenic Arabidopsis T2 generation plant groups (SiBRB-1 and SiBRB-2) and the wild type Arabidopsis plant group (WT) provided in the examples of the present invention;

FIG. 9 is a graph comparing the results of root fresh weight (mg) of the SiBRB transgenic Arabidopsis T2 generation plant groups (SiBRB-1 and SiBRB-2) and the wild type Arabidopsis plant group (WT) provided in the examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Molecular cloning is generally performed according to conventional conditions such as Sambrook et al: a Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or Draper et al (Blackwell scientific Press, 1988), or according to the conditions recommended by the manufacturer of the reagents used.

The method of cloning the sesame root size-related gene SiBRB described in the present invention is a method commonly used in the art. The extraction of Plant root RNA is a common molecular biology technique, various mature techniques are available for the extraction of mRNA, and kits (EASY spin Plus Plant RNA kit) are commercially available (from Aidlab Biotechnologies Co., Ltd.). The methods of enzyme digestion, ligation, inflorescence infection and the like used for constructing the vector and transferring the vector into a plant are also common techniques in the field. The plasmids involved therein (pCAMBIA1301), media for transfection (Agrobacterium tumefaciens LBA4404 and reagent components used such as sucrose, Kan, hygromycin, etc.) are commercially available.

Acquisition of sesame Gene SiBRB

The method for obtaining the sesame gene SiBRB comprises the following steps:

(1) according to the detection results of phenotype, geographical source and genetic diversity of drought-resistant related characters, a step-by-step sampling strategy is adopted from 7910 sesame germplasm resources at home and abroad stored in a national sesame middle-stage library, and 327 sesame samples with different drought resistances are selected for re-sequencing analysis;

(2) carrying out whole genome re-sequencing on 327 sesame samples with low coverage by using a2 x 76 double-end sequencing method by using an Illumina Hiseq2500 sequencing platform to obtain a genome sequence with 2.6 times coverage;

(3) combining 327 parts of root system and drought resistance (PEG mediated) related character data, genotype data and group structure of a germplasm resource group of a sesame sample under a water culture condition, performing whole genome association analysis on sesame related characters by adopting an EMMAX software package and a Peal program, and detecting 1 marker site which is positioned on 5024573 on the No. 15 linkage group and is obviously associated with the dry weight of the root system of the sesame and the total number of the roots;

(4) through sesame reference genome (http:// ocri-genomics.org/Sinbase/index. html) comparison and gene annotation analysis, the marker locus is found to fall inside the SIN-1025576 gene and exist at the position of 1713bp in the CDS sequence of the gene, and the gene has no homologous gene with Arabidopsis and other crops through homologous comparison, so the gene is a newly discovered sesame gene with unknown functions and is presumed to be related to sesame root development and named as SiBRB;

(5) primers were designed to amplify the entire CDS sequence of SiBRB gene using primer5.0, based on its CDS sequence (including modified bases) and the names:

SiBRB-F: 5'-gctttcgcgagctcggtaccatggaaatgagcatcccatt-3', as shown in SEQ ID NO. 3;

SiBRB-R: 5'-cgactctagaggatcctcatgacaagtgttggctgc-3', as shown in SEQ ID NO. 4;

(6) taking the root system of the sesame material G340 in the seedling stage, extracting total RNA of the root system, carrying out reverse transcription to generate cDNA, carrying out RT-PCR amplification by using the reversed cDNA as a template and using the primer SiBRB-F/SiBRB-R in the step S5, sequencing the amplified fragment, and obtaining the SiBRB gene sequence related to the development of the sesame root system, wherein the SiBRB gene sequence is shown as SEQ ID NO. 2.

Construction of SiBRB-transgenic Arabidopsis thaliana

1. Construction of recombinant expression vector of SiBRB gene

The sesame gene SiBRB cloned from the above example was ligated to pCAMBIA1301S (provided in this laboratory) using homologous recombination to construct a plant recombinant expression vector, which was designated pCAMBIA1301S-SiBRB (fig. 1). The specific operation is as follows:

(1) firstly, obtaining a linearized vector of pCAMBIA1301S by using a double enzyme digestion (BamHI and KpnI) (Takara) method, and then purifying the linearized vector by using an agarose gel electrophoresis and gel recovery kit (Tiangen Biochemical technology Co., Ltd.) to obtain a high-purity pCAMBIA 1301S;

(2) adding the target fragment DNA and a linearized vector pCAMBIA1301S into a centrifugal tube of 1.5ml according to the molar ratio of 3:1 for recombination reaction, uniformly mixing, placing at 37 ℃ for about 30min, adding 10 mu l of reaction solution into 50 mu l of DH5a competent cells, gently mixing by using a pipette, incubating on ice for 20min, thermally shocking in a water bath at 42 ℃ for 45 seconds, and rapidly placing on ice for cooling for 2 min;

(3) adding 300. mu.l LB liquid medium, and incubating at 37 ℃ for 45-60 min. Centrifuging at 5000rpm for 2min to collect thallus, discarding part of supernatant, re-suspending thallus with the rest culture medium, lightly and uniformly coating on LB solid culture medium containing Kan resistance with sterile coating rod, and culturing in 37 deg.C incubator for 16-24 hr;

(4) selecting a plurality of clones on the recombinant reaction conversion plate to carry out colony PCR identification, identifying as positive colonies, selecting corresponding single colonies to culture in a liquid LB culture medium containing Kan antibiotics at 37 ℃ and 200rpm for overnight, extracting plasmids or directly sequencing bacterial liquid, and identifying the carrier accuracy through enzyme digestion electrophoresis.

Any plant recombinant expression vector, transgenic cell line and recombinant strain containing the sesame gene SiBRB belong to the vectors for expressing the sesame gene SiBRB.

The recombinant expression vector containing the sesame gene SiBRB can be constructed by using the existing plant recombinant expression vector. The plant recombinant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin or other derivative plant recombinant expression vectors.

When the sesame gene SiBRB is used for constructing a plant recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin (Ubiquitin) gene promoter (pUbi) and the like, can be added in front of a transcription initiation nucleotide of the plant recombinant expression vector, and can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant recombinant expression vectors, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

2. Expression of

The specific expression steps in the embodiment of the invention are as follows:

the vector pCAMBIA1301S-SiBRB prepared in the examples was transferred to Agrobacterium tumefaciens LBA4404 (Shanghai Weidi Biotechnology Co., Ltd.), introduced into Arabidopsis thaliana plants, and expressed. The specific operation is as follows:

(1) the plant recombinant expression vector is transferred into agrobacterium LBA 4404:

adding 2 μ g plant recombinant expression vector pCAMBIA1301S-SiBRB into each 100 μ l LBA4404 Agrobacterium tumefaciens competent cell, stirring uniformly by hand to the bottom of the tube, standing on ice for 5min, adding liquid nitrogen for 5min, adding water bath at 37 deg.C for 5min, and ice-cooling for 5 min;

700. mu.l of LB liquid medium without antibiotics were added and cultured at 28 ℃ for 5 hours with shaking. And (3) centrifuging at 6000rpm for 1min to collect thalli, reserving about 100 mu l of supernatant, slightly blowing and beating the resuspended thalli, uniformly coating the thalli on an LB solid culture medium containing Kan and Rif, inversely placing the thalli in an incubator at 28 ℃ for 2 days, and picking a plurality of positive clones to simply verify the result by utilizing colony PCR.

(2) Plate culture of Arabidopsis thaliana:

1) counting a certain amount of arabidopsis seeds according to experiment requirements, and filling the arabidopsis seeds into a sterile 1.5mL centrifuge tube;

2) 1mL of 75% ethanol was added, the mixture was inverted and mixed, and the supernatant was discarded and repeated 1 time. Placing into a shaker at 37 deg.C and 200rpm, and shaking for 10min for surface sterilization;

3) discard 75% ethanol, add 1mL 95% ethanol, mix by inversion, discard the supernatant, and repeat 1 time. Adding 300-;

4) after the ethanol is volatilized, dibbling the arabidopsis seeds on a prepared flat plate by using toothpicks;

5) sealing the flat plate, performing vernalization at 4 ℃ for 48h under the dark condition, after vernalization, vertically culturing the flat plate in an illumination incubator, and transplanting after seedling emergence for one week;

6) the seedlings are planted in soil of a small pot by using tweezers, are firstly moisturized by using a preservative film for 24 hours, and are placed in a plant growth room for cultivation until the growth of arabidopsis thaliana is bolting (about one month) for transformation.

(3) Genetic transformation:

in order to facilitate identification and screening of transgenic plant cells or plants, a plant recombinant expression vector to be used may be processed, for example, by adding a gene (GUS gene, GFP gene, luciferase gene, etc.) expressing an enzyme or a luminescent compound which produces a color change in a plant, an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.) or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

The plant recombinant expression vector carrying the plant root development related protein coding gene SiBRB can be transformed into receptor plant cells or tissues by Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation and other conventional biological methods. The sesame gene SiBRB is introduced into the recipient plant by the plant recombinant expression vector.

The genetic transformation procedure for the SiBRB gene in this example is as follows:

1) activating agrobacterium: respectively adding 20 mu L of Rif and Kan (Sigma company) into 20mL of LB liquid culture medium, shaking uniformly, inoculating bacteria, and performing shaking activation at 28 ℃ and 220rpm for 8-10h to obtain activated bacteria liquid of agrobacterium;

2) and (3) agrobacterium tumefaciens enlarged culture: respectively adding 200 mul of Rif and Kan into 200mL of LB liquid culture medium, adding 5-10mL of activated bacterium liquid, shake-culturing at 28 ℃ and 220rpm for 14-16h until OD value is 1.6-2.0, centrifuging at 4500rpm for 10min, depositing thalli, discarding supernatant, and naturally drying;

3) adding 100mL of 5% sucrose solution into the precipitated thalli to resuspend the thalli, and blowing and beating the thalli uniformly by a pipette to resuspend the thalli;

4) adding the bacterial liquid in the centrifugal bottle into a plate, adding 100mL of 5% sucrose solution, adding 40 mu of LSilwet-L-77 (0.02%) before conversion, shaking the plate and uniformly mixing;

5) the Arabidopsis inflorescences were closed, immersed in a plate, and gently shaken for 15 s. After the conversion is finished, uniformly stirring the bacterial liquid;

6) sleeving the plants with black bags, keeping out of the sun and preserving moisture for 24 hours;

7) the transformation was repeated once more after one week.

(4) Screening positive plants of T1 generation

Seeds harvested from T0 generation of Arabidopsis are planted, seeds from T0 generation are disinfected, inoculated with MS screening culture medium containing 30mg/L hygromycin (25 mg/L of cefamycin is added for bacteriostasis) and cultured for 7-10 days under illumination at 22 ℃, and screened to obtain positive plants (plants with normal growth of seedlings and roots) from T1 generation (as shown in figure 2). The experiment obtains 6 positive strains, the positive seedlings are transplanted into soil, the film is uncovered after the soil is covered by a preservative film for 2 to 3 days, and then the seedlings grow normally. And (3) extracting DNA from leaves of the screened T1-generation positive plants, identifying that the leaves contain SiBRB genes by using a PCR method, performing molecular verification on target genes of transgenic plants (as shown in figure 3), and finally confirming that the genes are transferred into T1-generation positive plants.

(5) Positive detection of transgenic plant T2 generation

Carrying out single plant seed collection on the T1 generation positive plants to obtain T1 generation seeds, continuously carrying out hygromycin screening to obtain T2 generation positive plants (as shown in figure 4), transplanting and growing the obtained positive plants, extracting leaf genome DNA (deoxyribonucleic acid) to carry out PCR (polymerase chain reaction) molecular identification, and determining T2 generation positive plants (as shown in figure 5);

(6) quantitative expression verification of transgenic T2 positive plants

Taking a transgenic plant T2 generation positive plant and a young leaf of a wild type arabidopsis plant in the growth period, extracting total RNA of the leaf by using an RNA extraction kit (Beijing Adela biotechnology Co., Ltd.), then obtaining cDNA by using a reverse transcription kit (Nanjing NuoWei Zan biotechnology Co., Ltd.), taking respective cDNA as templates, and taking arabidopsis beta-actin as an internal reference (aF: 5'-CCCGCTATGTATGTCGCCA-3', shown as SEQ ID NO. 5; aR: 5'-AACCCTCGTAGATTGGCACAG-3', shown as SEQ ID NO. 6); the target gene quantitative primer sequence is as follows: BRBF: 5'-ATGGAAATGAGCATCCCATT-3', as shown in SEQ ID NO. 7; BRBR: 5'-TCATGACAAGTGTTGGCTGC-3', as shown in SEQ ID NO.8, qRT-PCR expression validation (qRT-PCR Mix: Nanjing Nodezam Biotech Co., Ltd.; Instrument: Roche LightCyclerR 480) was performed.

As shown in fig. 6, the results showed that, with non-transgenic wild arabidopsis thaliana as a control, the expression level of the sesame gene SiBRB was significantly increased in the leaves of the 3 arabidopsis transgenic lines tested, whereas no overexpression of the sesame gene SiBRB was detected in the leaves of the non-transgenic control arabidopsis thaliana, which indicated that the sesame gene SiBRB was transformed and inserted into the corresponding arabidopsis genome and overexpressed.

Root system assay of arabidopsis positive plants

Transgenic plants of the T2 generation were cultured vertically on square plastic culture dishes and measured at the seedling stage (10 days), and the dishes were scanned with EPSONV800, the results of which are shown in fig. 7. Total lateral root length was measured with ImageJ software (results are in fig. 8) and root fresh weight was weighed (results are in fig. 9).

The SiBRB transgenic Arabidopsis T2 generation plant group in FIG. 7 is compared with the wild Arabidopsis plant group, which shows that the root system of the wild Arabidopsis plant group grows more vigorously. FIG. 8 shows that the total lateral root length is significantly lower for both the SiBRB-1 and SiBRB-2 groups than for the WT group. FIG. 9 shows that both the SiBRB-1 and SiBRB-2 groups had significantly lower root fresh weights than the WT group. Therefore, the total lateral root length and the fresh root weight of the Arabidopsis thaliana strain transformed with the SiBRB gene obtained in the research are obviously lower than those of the wild Arabidopsis thaliana control, and the overexpression of the sesame gene SiBRB is shown to weaken the root system development of a receptor plant.

In conclusion, the sesame gene SiBRB for regulating and controlling the growth and development of plant roots is cloned and utilized in production, so that a more characteristic and effective gene resource is provided for the research on the regulation and control of the root cap ratio of main crops and the efficient absorption and utilization of nutrients, the sesame gene SiBRB plays an important role in the research on the improvement of the root cap ratio of plants through genetic engineering and the efficient absorption and utilization of nutrients, and has important practical value and direct economic benefit.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> institute of oil crop of academy of agricultural sciences of China

Application of sesame protein SiBRB in regulation and control of plant root system development

<141>2020-06-05

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